1. Field of the Invention
The present invention relates generally to measuring the forces a cell exerts on its surroundings, and in particular, to a NEMS (nano-electromechanical system) sensor for cell force application and measurement.
2. Description of the Related Art
(Note: This application references a number of different publications as indicated throughout the specification by reference numbers enclosed in brackets, e.g., [x]. A list of these different publications ordered according to these reference numbers can be found below in the section entitled “References.” Each of these publications is incorporated by reference herein.)
The ability to measure the forces a cell exerts on its surrounding may be useful in a variety of technological fields. However, the prior art has failed to provide a capability to measure such forces with a sufficient and useful resolution. To better understand such deficiencies, a description of prior art systems that could benefit from such measurements may be useful.
Mechanical cues in the form of ECM (extra cellular matrix) compliance have been shown to affect a wide range of physiological processes including stem cell differentiation [1], vascular development [2], fibroblast motility [3], glioblastoma metastasis [4] and breast cancer tumor progression including invasion and metastasis [5-7]. However, despite excellent and creative work by a number of research groups, understanding of these processes, generally referred to as mechanotransduction, remains limited to a conceptual framework supported by important but sparse instances of specific molecular information [8].
Mechanotransduction remains vaguely understood because tools that quantitatively probe the cell-ECM force balance are lacking. Although, significant progress has been made in the last 10 years as the biological community has turned its attention to these problems [9-11], large and critical areas of experiment space remain inaccessible. Of particular need are tools that directly measure the cell-ECM force balance with sufficient resolution to observe the initial ECM compliance sensing events and sufficient dynamic range to track the evolution of those events into whole cell phenotype and genotype changes, such as metastasis. Furthermore, it is insufficient to merely be able to access larger portions of the relevant experiment space, rather scalable tools that provide robust and repeatable quantitative data are needed in order to identify the critical proteins and regulatory pathways in each specific system.
In addition, cellular contractility—the internal generation of force or tension by a cell—has emerged as a critical regulator of a wide range of processes in organism development. Successful embryogenesis depends upon proper maintenance of tension and stiffness within the embryo. Tension directs stem cell differentiation and cell proliferation. Forces appear to constrain the spatial organization of cells in the formation of tissues and organs. Cancer development and metastasis also depend on internal tension. Contractility is primarily driven by actomyosin force generation, which is well understood as a standalone force generating unit. However, inside a cell, the basic actomyosin unit forms a variety of distinct force generating structures or “modules” such as cortical branched networks, transverse arcs and ventral stress fibers each of which generate distinct forces and interactions with the other force modules. The specifics of force generation and feedback by the various modules are at best poorly understood. Instrumentation has been a major hindrance. Tools that can repeatedly both measure forces and mechanically perturb sub-cellular structures with near single molecule resolution and whole cell dynamic range are needed to develop a complete and quantitative understanding of the actin cytoskeleton.
Stated another way, force production in cells has been a topic of interest for some time[12][13], but interest has increased significantly since recent demonstrations that cell contraction was involved in and even responsible for tumor formation[6-7][14-16], embryogenesis[2], stem cell differentiation[1] and organ development[17-18]. Numerous studies have been dedicated to investigating the consequences of contractility on physiology (intra-cellular organization[19], cell polarity[20], cell migration[21], cell growth rate[22], cell division orientation[23], cell positioning[24], tissue cohesion[25], tissue stiffening[5]), usually in response to RhoA activation. However, few studies have focused on the origin and exact mechanism of force production. This lack of analysis is principally due to technological limitations. Indeed, most existing force measurement methods only access the global net force exerted by the cell. They are unable to measure local intra-cellular forces or to identify which intra-cellular structure is responsible for which part of the global force produced[9-11]. However, cell polarity, cell migration or tissue stiffening, for example, most likely do not depend on contraction of the same intra-cellular structures. Stress fibers are not the only cell structure supporting mechanical loads in cells. Various force production modules exist and their respective contractile and mechanical characteristics surely have differing impacts on cell behavior. Thus, it would be beneficial to distinguish the exact role of each force production module in order to understand the physiological consequences of their specific regulation and deregulation across the broad range of effects of intra-cellular contraction.
In view of the above, what is needed is a device that is useful for measuring the forces a cell exerts on its surroundings.